Zika virus recombinant adenovirus shuttle plasmid expressing e protein mutant, adenovirus plasmid, virus and preparation method and application thereof

Abstract

The application discloses a recombinant adenovirus shuttle plasmid and a preparation method thereof, a recombinant adenovirus and application thereof, and a Zika virus recombinant adenovirus plasmid, and belongs to the field of biological medicines. The Zika virus recombinant adenovirus shuttle plasmid provided by the application takes pShuttle as a carrier, carries a Zika virus E protein mutant gene, and the 5' end of the Zika virus E protein mutant gene is connected with a signal peptide gene; the 5' end of the signal peptide gene is connected with a Kozak sequence; and the 5' end of the Kozak sequence is connected with an enzyme cutting site 1. The recombinant shuttle plasmid, the recombinant plasmid and the recombinant adenovirus provided by the application can be applied to the preparation of a Zika virus adenovirus vaccine, and have wide development and application prospects.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a Zika virus recombinant adenovirus shuttle plasmid, an adenovirus plasmid, a virus, a method for preparing the same, and its applications; more specifically, to a Zika virus recombinant adenovirus shuttle plasmid and its preparation method, a Zika virus recombinant adenovirus and its applications, and a Zika virus recombinant adenovirus plasmid. Background Technology

[0002] Zika fever is an acute infectious disease caused by the Zika virus (ZIKV), primarily transmitted by Aedes albopictus and Aedes aegypti mosquitoes. It is a significant zoonotic disease in tropical and subtropical regions, having caused an outbreak in South America from 2015 to 2016, infecting millions. Currently, nearly 100 countries and regions worldwide have experienced Zika virus outbreaks or importations. Zika virus infection in pregnant women can cause microcephaly in the fetus, while infection in adults can lead to Guillain-Barré syndrome. Current prevention and treatment methods for Zika fever mainly include vaccination and drug therapy, but many challenges remain in prevention and control. For example, the confirmation of Zika virus infection often relies on PCR and ELISA detection, which are cumbersome procedures; antibodies induced by Zika virus infection or candidate vaccination can exacerbate infections with other flaviviruses such as Japanese encephalitis virus (JEV) and dengue virus (DENV) through antibody-dependent enhancement (ADE) in vitro and in vivo. Therefore, finding a new method for the prevention and detection of Zika virus is imperative. Summary of the Invention

[0003] The problem this invention aims to solve is how to improve the safety of methods for preventing or detecting Zika virus.

[0004] To address the aforementioned problems, this invention provides a recombinant adenovirus shuttle plasmid and its preparation method, a recombinant adenovirus and its applications, and a Zika virus recombinant adenovirus plasmid. The Zika virus recombinant adenovirus shuttle plasmid carries the Zika virus wild-type E protein gene or a Zika virus E protein mutant gene. The amino acid sequence encoded by the Zika virus wild-type E protein gene is shown in SEQ ID No. 1, and the amino acid sequence encoded by the Zika virus E protein mutant gene is shown in SEQ ID No. 3.

[0005] The E protein is the main structural protein of the Zika virus, a hydrophobic glycoprotein closely related to Zika virus infection and pathogenesis. It exhibits high biological conservation and is a commonly used target protein for Zika virus detection and treatment. Vaccines developed using the E protein gene are generally able to protect animals from lethal doses of Zika virus challenge; therefore, the E protein gene is an excellent vaccine target. Adenovirus vectors are a commonly used genome modification technique that utilizes adenoviruses as vectors. Adenoviruses are double-stranded DNA viruses with high gene transfer efficiency, capable of expressing or integrating exogenous target genes into host cells. Adenovirus vectors are typically constructed based on the wild-type adenovirus genome backbone, by disrupting the adenovirus's replication and encapsulation capabilities while inserting the sequence of the exogenous gene to be expressed. The insertion site for the exogenous gene is usually the region where the original adenovirus's E1 / E3 sequences have been removed. Adenovirus vectors possess the following characteristics: 1. They can infect various types of mammalian cells, including non-dividing cells, slow-proliferating cells, and normal cells; they can stably exist in human cells; 2. They can stably express exogenous genes without affecting host gene expression and function; 3. They exhibit lower immunogenicity and cytotoxicity compared to other gene transfer vectors; 4. They are easy to prepare on a large scale and extract with high purity. Therefore, adenovirus vectors have broad application prospects in gene therapy, gene vaccines, and gene research.

[0006] Preferably, the nucleotide sequence of the wild-type Zika virus E protein gene is shown in SEQ ID No. 2, and the nucleotide sequence of the Zika virus E protein mutant gene is shown in SEQ ID No. 4.

[0007] Preferably, the 5' end of the Zika virus wild-type E protein gene and the Zika virus E protein mutant gene is linked to a signal peptide gene, the 5' end of the signal peptide gene is linked to a Kozak sequence, the 5' end of the Kozak sequence is linked to an enzyme cleavage site, the amino acid sequence encoded by the signal peptide gene is shown in SEQ ID No. 5, the nucleotide sequence of the signal peptide gene is shown in SEQ ID No. 6, the specific nucleotide sequence of the Kozak sequence is gccacc, and the specific nucleotide sequence of the enzyme cleavage site is gaattc.

[0008] Preferably, the 3' end of the Zika virus wild-type E protein gene and the Zika virus E protein mutant gene is connected to an enzyme cleavage site II, and the specific nucleotide sequence of the enzyme cleavage site II is: ctcgag.

[0009] Preferably, the vector for the Zika virus recombinant adenovirus shuttle plasmid is pShuttle.

[0010] Furthermore, a second aspect of the present invention provides a method for preparing the aforementioned Zika virus recombinant adenovirus shuttle plasmid, comprising the following steps: S1: The pShuttle plasmid, the wild-type Zika virus E protein gene, or the Zika virus E protein mutant gene were double-digested using the restriction endonucleases EcoRI and Xhol. S2: Perform 0.8% agarose gel electrophoresis on the enzyme digestion products of step S1, and cut out the bands of the target gene and pShuttle respectively. Use an agarose gel purification and recovery kit to recover pShuttle and the target gene, Zika virus wild-type E protein gene or Zika virus E protein mutant gene. S3: Use T4 DNA ligase to ligate pShuttle with the wild-type Zika virus E protein gene or the Zika virus E protein mutant gene; S4: Add the reaction system after step S3 to Stbl3 competent cells, gently mix, incubate on ice for 30 min, incubate in water at 42℃ for 90 s, quickly transfer to ice and incubate on ice for 2 min, add antibiotic-free LB medium to the cells, and incubate with shaking at 180 rpm / 37℃ for 1 h. S5: Centrifuge, remove the supernatant, mix the remaining bacterial culture and inoculate it onto LB agar medium containing 50 μg / mL kanamycin, incubate at 37°C upright until the liquid is absorbed, then invert and incubate at 37°C overnight. S6: Select white bacterial clones and add them to 10 mL of LB medium (containing 50 μg / mL kanamycin), and incubate at 220 rpm / 37℃ with shaking for 18 h; S7: Extract recombinant plasmids from bacterial cultures using a plasmid mini-extraction kit and determine the plasmid concentration.

[0011] Furthermore, a third aspect of the present invention provides a Zika virus recombinant adenovirus plasmid, wherein the plasmid carries an exogenous Zika virus wild-type E protein gene or a Zika virus E protein mutant gene, obtained by transforming the Zika virus recombinant adenovirus shuttle plasmid according to any one of claims 1 to 5 into human adenovirus type 5 genome backbone plasmid pAdEasy-1 into Escherichia coli BJ5183 for homologous recombination and then screening.

[0012] Furthermore, it was obtained by transfecting the aforementioned Zika virus recombinant adenovirus plasmid into a host cell.

[0013] Furthermore, a fifth aspect of the present invention provides the application of the aforementioned Zika virus recombinant adenovirus in the preparation of a Zika virus recombinant adenovirus vaccine.

[0014] Adenovirus vector vaccines are a novel type of vaccine that uses replication-defective adenovirus as a vector. They have advantages such as high transduction efficiency and high safety. This invention uses the Zika virus mutant E protein (E protein mutant) as the target site, adenovirus vector as raw material, and AdEasy as the recombinant system to prepare a Zika virus recombinant adenovirus vaccine.

[0015] Preferably, the application includes the following steps: incorporating Zika virus recombinant adenovirus Ad5-ZIKV-E... WT and / or Ad5-ZIKV-E mut Zika virus recombinant adenovirus vaccine was prepared by continuously passaged after inoculation into host cells and stabilizing the transcription and expression of recombinant adenovirus E protein in host cells.

[0016] The beneficial effects of this invention are as follows: This invention overcomes the shortcomings of existing technologies and provides a Zika virus recombinant adenovirus, shuttle vector, plasmid, and its application in vaccine preparation. The plasmid carrying the target gene is double-digested using restriction endonucleases EcoRI and Xhol to obtain a linearized plasmid. The digestion products are then subjected to 0.8% agarose gel electrophoresis, and the band containing the target gene and pShuttle is excised and recovered. T4 DNA ligase is used to ligate pShuttle to the target gene, yielding a recombinant shuttle plasmid. The recombinant shuttle plasmid is co-transformed with the adenovirus vector system backbone vector pAdEasy-1 into *E. coli* strain BJ5183 to obtain a recombinant adenovirus plasmid. The recombinant adenovirus plasmid is transfected into 293T cells to obtain recombinant adenovirus. Western blot analysis confirms the role of the ZIKV-E protein in the recombinant adenovirus Ad5-ZIKV-E. WT Ad5-ZIKV-E mut Stable expression in [the target gene]; after immunizing mice with recombinant adenovirus, the target gene can be effectively transferred into the body and expressed, and can effectively stimulate the body to produce an immune response. Compared with Ad5-ZIKV-E WT Ad5-ZIKV-E mut The antibodies induced in immunized mice showed no significant difference in their ability to neutralize Zika virus in vivo and in vitro. However, their ability to cross-recognize Japanese encephalitis virus (JEV)-E protein and dengue virus (DENV)-E protein was significantly reduced, and the antibody-dependent enhancement (ADE) effect against JEV and DENV infection was also significantly reduced in vivo and in vitro. Therefore, the recombinant adenovirus vaccine of this invention is safe and effective, can stimulate the body to produce ZIKV neutralizing antibodies, and significantly reduces the ADE effect against JEV and DENV infection, thus showing broad development and application prospects. Attached Figure Description

[0017] Figure 1 shows the experimental results of Western Blot in Example 5 of the specific embodiments of the present invention; Figure 2 shows the ELISA detection results in Example 6 of the specific embodiments of the present invention; Figure 3 shows the experimental results of the Zika virus neutralization experiment in Example 7 of the specific embodiments of the present invention; Figure 4 shows the experimental results of the in vitro ADE experiment in Example 8 of the specific embodiments of the present invention; Figure 5 shows the experimental results of the neutralizing effect of adoptive immune serum on ZIKV infection in mice in Example 9 of the specific embodiments of the present invention; Figure 6 shows the experimental results of the enhancing effect of adoptive immune serum on DENV2 infection in mice in Example 10 of the specific embodiments of the present invention; Figure 7 shows the experimental results of the enhancement effect of antibodies obtained from the mother in suckling mice on JEV infection in Example 11 of the specific embodiments of the present invention. Detailed Implementation

[0018] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, specific embodiments of the present invention are described in detail below. It should be noted that the following embodiments are only used to illustrate the implementation methods and typical parameters of the present invention, and are not intended to limit the parameter range described in the present invention. Reasonable variations derived therefrom are still within the protection scope of the claims of the present invention.

[0019] It should be noted that the endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0020] In a specific embodiment of the present invention, the Zika virus wild-type E protein gene and ZIKV-E WT Having the same meaning, the Zika virus E protein mutant gene is similar to ZIKV-E. mut They have the same meaning; adenovirus and Ad5 have the same meaning.

[0021] The present invention provides a Zika virus recombinant adenovirus shuttle plasmid and its preparation method. The Zika virus recombinant adenovirus shuttle plasmid provided by the present invention carries the Zika virus wild-type E protein gene (encoding the amino acid sequence shown in SEQ ID No. 1 and the nucleotide sequence shown in SEQ ID No. 2) or the Zika virus E protein mutant gene (encoding the amino acid sequence shown in SEQ ID No. 3 and the nucleotide sequence shown in SEQ ID No. 4).

[0022] In the Zika virus recombinant adenovirus shuttle plasmid provided in a specific embodiment of the present invention, the plasmid vector is pShuttle. The 5' end of the Zika virus wild-type E protein gene or the Zika virus E protein mutant gene is linked to a signal peptide gene. The 5' end of the signal peptide gene is linked to a Kozak sequence, and the 5' end of the Kozak sequence is linked to restriction enzyme site 1. The amino acid sequence encoded by the signal peptide gene is shown in SEQ ID No. 5, and the nucleotide sequence of the signal peptide gene is shown in SEQ ID No. 6. The specific nucleotide sequence of the Kozak sequence is gccacc, and the specific nucleotide sequence of restriction enzyme site 1 is gaattc. The 3' end of the Zika virus wild-type E protein gene or the Zika virus E protein mutant gene is linked to a second restriction enzyme site, and the specific nucleotide sequence of restriction enzyme site 2 is ctcgag.

[0023] The method for preparing the aforementioned Zika virus recombinant adenovirus shuttle plasmid provided in the specific embodiments of the present invention includes the following steps: S1: The pShuttle plasmid, the wild-type Zika virus E protein gene, or the Zika virus E protein mutant gene were double-digested using restriction endonucleases EcoRI and Xhol. S2: Perform 0.8% agarose gel electrophoresis on the enzyme digestion products of step S1, and cut out the bands of the target gene and pShuttle respectively. Use an agarose gel purification and recovery kit to recover pShuttle and the target gene, Zika virus wild-type E protein gene or Zika virus E protein mutant gene. S3: Use T4 DNA ligase to ligate pShuttle with the wild-type Zika virus E protein gene or the Zika virus E protein mutant gene; S4: Add the reaction system after step S3 to Stbl3 competent cells, gently mix, incubate on ice for 30 min, incubate in water at 42℃ for 90 s, quickly transfer to ice and incubate on ice for 2 min, add antibiotic-free LB medium to the cells, and incubate with shaking at 180 rpm / 37℃ for 1 h. S5: Centrifuge, remove the supernatant, mix the remaining bacterial culture and inoculate it onto LB agar medium containing 50 μg / mL kanamycin. Incubate at 37°C upright until the liquid is absorbed, then invert and incubate at 37°C overnight. S6: Select white bacterial clones and add them to 10 mL of LB medium (containing 50 μg / mL kanamycin), and incubate at 220 rpm / 37℃ with shaking for 18 h; S7: Extract recombinant plasmids from bacterial cultures using a plasmid mini-extraction kit and determine the plasmid concentration.

[0024] This invention also provides a Zika virus recombinant adenovirus plasmid, which is prepared through the following steps: The aforementioned Zika virus recombinant adenovirus shuttle plasmid and the human adenovirus type 5 genome backbone plasmid pAdEasy-1 were transformed into E. coli BJ5183 for homologous recombination. After screening, the following Zika virus recombinant adenovirus plasmid containing the exogenous Zika virus wild-type E protein gene or the Zika virus E protein mutant gene was obtained: Ad5-ZIKV-E WT Or Ad5-ZIKV-E mut .

[0025] The present invention also provides a recombinant Zika virus adenovirus, the preparation steps of which are as follows: The aforementioned recombinant Zika virus adenovirus plasmid is transfected into host cells to obtain the recombinant Zika virus adenovirus: Ad5-ZIKV-E. WT and / or Ad5-ZIKV-E mut .

[0026] The present invention also provides an application of the aforementioned Zika virus recombinant adenovirus in the preparation of a Zika virus recombinant adenovirus vaccine.

[0027] Example 1. Amplification and extraction of shuttle plasmid pShuttle 1.1. Centrifuge 0.5 μg PShuttle plasmid dry powder tube at 5000 rpm for 1 min, and add 20 μL ddH2O to dissolve the plasmid; 1.2. Take one 100 μL of Stbl3 competent bacteria and thaw it on ice for 10 min to allow it to thaw completely. Add 2 μL (50 ng) of plasmid, gently mix, and then incubate on ice for 30 min. Insert a float and heat shock it in a 42°C water bath for 90 s, then remove it and incubate on ice for 2 min. 1.3. Add 900 μL of antibiotic-free LB medium and incubate with shaking at 180 rpm / 37°C for 45 min; 1.4. Centrifuge at 6000 rpm for 5 min, discard 950 μL of supernatant, resuspend the bacterial pellet in the remaining liquid, and streak the bacteria onto the surface of LB agar plates containing kanamycin and streptomycin (both 50 μg / mL, spread 30 min in advance) using an inoculation loop; 1.5. Incubate the plate upright for 1 h, then invert it and incubate at 37°C for 14 h; 1.6. Pick a single colony and add it to 20 mL of LB medium containing antibiotics (50 μg / mL kanamycin, 50 μg / mL streptomycin). Incubate at 220 rpm / 37 °C for 14 h with shaking. Collect 10 mL of the culture and extract plasmids.

[0028] 1.7. Take the above bacterial culture, centrifuge at 12000 rpm for 1 min, and try to collect the supernatant (if there is a large amount of bacterial culture, the bacterial cells can be collected into a centrifuge tube by multiple centrifugations). 1.8. Add 250 μL of Solution I (with added RNase A) to the centrifuge tube containing the bacterial pellet, and thoroughly suspend the bacterial pellet using a pipette or vortex mixer; 1.9. Add 250 μL of Solution II to the centrifuge tube and gently invert it 6-8 times to ensure complete lysis of the bacterial cells; 1.10. Add 350 μL of Solution III to the centrifuge tube, and immediately gently invert it 6-8 times to mix thoroughly. A white flocculent precipitate will appear. Centrifuge at 12000 rpm for 10 min. Carefully transfer the supernatant to another clean centrifuge tube using a pipette, trying not to aspirate the precipitate. 1.11. Add the supernatant obtained in the previous step to the adsorption column (add the adsorption column to the collection tube), let it stand at room temperature for 2 min, centrifuge at 12000 rpm for 1 min, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube. 1.12. Add 600 μL of wash solution I (with anhydrous ethanol added) to the adsorption column, centrifuge at 12000 rpm for 1 min, discard the waste liquid, and place the adsorption column into the collection tube; 1.13. Add 700 μL of wash solution II (with anhydrous ethanol added) to the adsorption column, centrifuge at 12000 rpm for 1 min, discard the waste liquid, and place the adsorption column into the collection tube; 1.14. Add 500 μL of wash solution II to the adsorption column, centrifuge at 12000 rpm for 1 min, discard the waste liquid, and place the adsorption column into the collection tube; 1.15. Centrifuge at 12000 rpm for 2 min, then place the adsorption column open at room temperature or in a 50°C incubator for several minutes to remove the washing solution that participated in the adsorption column. 1.16. Place the adsorption column into a clean centrifuge tube, add 50-200 μL of preheated elution buffer (preheated in a 65°C water bath) dropwise to the center of the adsorption membrane, incubate at room temperature for 2 min, and centrifuge at 12000 rpm for 1 min. 1.17. To increase the plasmid recovery efficiency, the obtained eluent can be added back into the adsorption column, incubated at room temperature for 2 min, and centrifuged at 12000 rpm for 1 min. 1.18. The concentration of the extracted plasmid was measured using a spectrophotometer.

[0029] Example 2. Construction of the recombinant shuttle plasmid pShuttle 2.1. Determine the concentration of plasmids containing the target gene and the pShuttle plasmid. 2.2. The plasmid containing the exogenous gene and the shuttle plasmid pShuttle were double-digested in two sterile 1.5 mL EP tubes, as shown in Table 1; Table 1. EcoRI and Xhol double enzyme digestion reaction system reagent components Volume / Dosage plasmid 1~2 μg 10 × FlyCut Buffer 5 μL EcoRI 1 μL Xhol 1 μL RNase-free water Add to 50 μL Mix well, incubate in a 37°C water bath for 5-15 minutes, then inactivate by heating at 65°C for 20 minutes. 2.3. Perform 1% agarose gel electrophoresis on 50 μL of the enzyme digestion product. Cut out the bands containing the exogenous gene and PSHouter separately, and put them into two 1.5 mL EP tubes. Weigh them. If the gel weight is 100 mg, it can be regarded as 100 μL (100 mg = 100 μL), and so on. 2.4. Add 3 times the volume of GSB solution to the tube and melt the gel in a 55°C water bath for 6-10 min, mixing intermittently (2-3 min) to ensure the gel is completely melted. After the gel is completely melted, observe the color of the solution. If the color is purple, add an appropriate amount of 3 M sodium acetate (pH 5.2) to adjust the color to be the same as the color of GSB (yellow). 2.5. Cool the gel solution to room temperature, add it to a centrifuge column, let it stand for 1 min, centrifuge at 10000 g for 1 min, and discard the column liquid; 2.6. Add 650 μL of WB solution, centrifuge at 10000 g for 1 min, and discard the column liquid; 2.7. Centrifuge at 10000 g for 1-2 min to completely remove residual white blood cells (WB); 2.8. Place the centrifuge column in a clean centrifuge tube, open the cap and let it stand for 1 min to allow the residual ethanol to evaporate completely. Add 30-50 μL of deionized water (pH>7.0) to the center of the column (preheat the deionized water in a 60-70°C water bath) and let it stand at room temperature for 1 min. 2.9. Centrifuge at 10000 g for 1 min to elute DNA, and store the eluted DNA at -20°C; 2.10. Prepare the reagents according to Table 2 and carry out the ligation reaction; Table 2. Shuttle plasmid ligation reaction system reagent components Volume / Dosage pShuttle 0.1 μg exogenous genes 0.3 μg T4 DNA ligase (5 U / μL) 1 μL Ligase buffer (5×) 4 μL ATP (20 mmol / L) 1 μL RNase-free water Add to 20 μL The reaction mixture was incubated overnight at 16°C.

[0030] 2.11. Remove Stbl3 competent cells from the -80°C freezer and quickly place them in an ice box to thaw. Add 10 μL of ligation product to an EP tube containing 50 μL of competent cells. Incubate on ice for 30 min, then in a 42°C water bath for 90 s. Gently and quickly transfer the tube to ice and incubate on ice for 2 min. Do not shake the EP tube during this process. Add 500 μL of antibiotic-free LB medium to the centrifuge tube and incubate with shaking at 180 rpm / 37°C for 60 min. 2.12. Centrifuge the EP tube at 3000 rpm for 5 min, discard 450 µl of supernatant, and inoculate the remaining 100 µl of bacterial culture onto LB agar medium containing 50 μg / mL kanamycin and 50 μg / mL streptomycin. Spread evenly and incubate at 37°C upright until the liquid is absorbed. Then invert and incubate at 37°C overnight. 2.13. Pick white colonies from the surface of LB agar medium and add them to 2 mL of LB broth (containing 50 μg / mL kanamycin), and incubate with shaking at 220 rpm / 37°C for 18 h; 2.14. Plasmid DNA was extracted from bacterial cultures using a plasmid extraction kit. The recombinant shuttle plasmid was digested with the restriction endonuclease PmeI, and 5 μL of the digest was subjected to agarose gel electrophoresis to confirm complete plasmid digestion, ensuring the acquisition of a linearized shuttle plasmid.

[0031] Example 3. Construction of recombinant adenovirus plasmid 3.1. Treatment of electrocution cup: Soak in 75% alcohol for 30 min, clean with sterile ddH2O 5-10 times, and irradiate with ultraviolet light and ventilation in a clean bench for 30 min; 3.2. Add 1-5 µl of linearized shuttle plasmid to a pre-cooled EP tube containing 50 µl of BJ5183 (padeasy-1) competent cells, mix well, and cool on ice; 3.3. Quickly transfer the mixture to a pre-cooled electrostatic precipitator, wipe the surface of the electrostatic precipitator dry, and quickly place it into the electrostatic precipitator for electric shock (1250-1500V / mm, 5ms). 3.4. After electroporation, quickly transfer the electroporation cup to ice, immediately add 1 mL of antibiotic-free LB medium, quickly and gently resuspend the cells with a pipette and transfer them into an EP tube, and incubate with shaking at 225 rpm / 30℃ for 40 min. 3.5. Take an appropriate volume of the electroporated cell transformant and spread it on an agar plate containing 50 µg / mL kanamycin. Incubate upright for 1 h, then invert at 37°C for 16-20 h. 3.6. The next day, pick the colonies that have grown on the plate (select the smallest white colonies), inoculate them into 3 mL of LB medium containing 50 µg / mL kanamycin, and incubate at 37℃ for 10-15 h; 3.7. Plasmids were extracted using a plasmid extraction kit and subjected to 0.8% agarose gel electrophoresis. Large plasmids were considered possible positive clones and were further identified by PacI restriction enzyme digestion. 3.8. Digest with PacI and perform 0.8% agarose gel electrophoresis. If the electrophoresis shows one large fragment (approximately 30 kb) and one small fragment (approximately 3.0 kb or 4.5 kb), it is identified as the recombinant adenovirus plasmid: Ad5-JEV-E. WT and Ad5-JEV-E mut ; 3.9. The recombinant adenovirus plasmid was transformed into DH5α or Stbl3 competent cells to amplify the bacteria. The plasmid was then extracted and identified by Pac I single enzyme digestion and EcoRI / Xho I double enzyme digestion, finally obtaining a large number of recombinant adenovirus plasmids.

[0032] Example 4. Preparation of recombinant adenovirus 4.1. PacI was used to target the recombinant adenovirus plasmid Ad5-ZIKV-E. WT and Ad5-ZIKV-E mutLinearized plasmids were obtained by single enzyme digestion. The enzyme digestion reaction system is shown in Table 3. After preparing the enzyme digestion system according to the components in Table 3, the mixture was mixed by pipetting and aspiration. The liquid on the tube wall was collected to the bottom of the tube by instant centrifugation. The tube was then incubated at 37℃ for 30 min. After the enzyme digestion was completed, 5 μL of the enzyme digestion product was electrophoresed to identify whether the plasmid was cleaved. The remaining part was frozen at -20℃ for later use. Table 3 PacI single enzyme digestion reaction system reagent components Volume / Dosage <![CDATA[Ad5-ZIKV-E WT Or Ad5-ZIKV-E mut ]]> 2 μg 10 × NE Buffer 5 μL PacI 1 μL RNase-free water Add to 50 μL 4.2. The day before transfection, the cultured 293T cells were seeded on a 10 cm culture dish, with the cell growth density at transfection being between 70% and 80%; 4.3. Four hours before transfection, aspirate the original culture medium from the culture dish using a pipette and replace it with MEM incomplete culture medium; add 30 mg of linearized Ad5-ZIKV-E. WT Or Ad5-ZIKV-E mut The plasmid was dissolved in physiological saline (total volume 0.5 mL), and then 60 mL of PEI transfection reagent was added. The mixture was incubated at room temperature for 20 min. The transfected plasmid was then gently added dropwise into 293T cells. 4.4. Four hours after transfection, discard the cell supernatant, add 8 mL of MEM complete culture medium, and incubate in a 37℃ / 5%CO2 incubator; 4.5. Once obvious cytopathic effects appear in the cells, collect the cells and supernatant, and repeatedly freeze-thaw between -80℃ and 37℃ four times. After centrifugation at 4000 rpm for 10 min, collect the viral supernatant and inoculate it into the 293T cells seeded the day before. Repeat the above three rounds, collecting cytopathic cells and supernatant, repeatedly freezing and thawing between -80℃ and 37℃ four times, and collecting the viral supernatant after centrifugation at 4000 rpm for 10 min. 4.6. Centrifuge the viral supernatant at 10,000 rpm for 30 min, collect the supernatant, and ultracentrifuge the supernatant at 50,000 rpm for 2 h. Resuspend the viral precipitate in PBS. Measure the absorbance of the sample at 260 nm and 280 nm wavelengths using a UV spectrophotometer. PBS serves as a blank control. If the OD260 value is between 0.1 and 1, and the OD260 / OD280 ratio is between 1.2 and 1.4, then the recombinant adenovirus particle (VP) count / mL = OD260 × 10¹². If the OD260 value exceeds 1, appropriate dilution is required; the recombinant adenovirus particle (VP) count / mL = OD260 × dilution factor × 10¹².

[0033] Example 5. Recombinant adenovirus infecting cells 5.1. The day before infection, cultured 293T cells were seeded into 24-well cell culture plates, with the cell growth density at infection being between 70% and 80% as ideal; 5.2. The final recombinant adenovirus Ad5-ZIKV-E WT Or Ad5-ZIKV-E mut Add 293T cells (MOI=1), infect at 37℃ for 2 h, discard the virus solution, add MEM complete culture medium, and incubate at 37℃ / 5%CO2 for 2 days; 5.3. Collect the cell supernatant into a 1.5 mL EP tube, centrifuge at 8000 rpm for 5 min, and transfer the supernatant into a new 1.5 mL EP tube for Western blotting experiments.

[0034] Example 6. Western Blot assay for detecting the target protein 6.1. Prepare 15% SDS-PAGE gel; 6.2. Take 48 μL of the cell supernatant and add 12 μL of 5× protein loading buffer. Mix well and boil at 100℃ for 5 min. Add 20 μL to the sample well. Electrophoresis at 100 V for about 40 min. When the sample has migrated between the upper and lower gel layers, increase the voltage to 120 V and electrophoresis for another 120 min. 6.3. After electrophoresis, cut PVDF membranes to the size of the gel, activate the membranes in methanol solution for 1 min, and then transfer them to 1× electroporation buffer for 5 min to equilibrate. Prepare a sandwich clamp in the following order: sponge, filter paper, gel, PVDF membrane, filter paper, sponge. Place the sandwich clamp into the electrophoresis tank with the membrane facing the positive electrode and the gel facing the negative electrode. Add 1× electroporation buffer to cover the sponge, place the electrophoresis tank in an ice-water mixture, and electrophoresis at 100 V for 2 h. 6.4. After electroporation, remove the PVDF membrane, wash it once with PBS, place it on a shaker, and block it with 5% skim milk powder at room temperature for 3.5 h (70 rpm). 6.5. After blocking, wash the membrane with 0.1% PBST (PBS containing 0.1% Tween) for 5 min each time, for a total of 3 times. Add the primary antibody (ZIKV-E410 protein-immunized mouse serum): 0.1% PBST = 1:2000 to the primary antibody dilution buffer and incubate overnight at 4°C. 6.6. Discard the primary antibody mixture and wash the membrane with 0.1% PBST for 5 min each time, for a total of 5 times. Add secondary antibody (HRP-labeled goat anti-mouse IgG monoclonal antibody): 0.1% PBST = 1:5000 to the secondary antibody dilution buffer and incubate at room temperature for 1 h; 6.7. Discard the secondary antibody mixture, wash the membrane with 0.1% PBST for 5 minutes each time, for a total of 5 times. Mix ECL chemiluminescence solution A and B in a 1:1 ratio and add it evenly to the PVDF membrane. Incubate for 1 minute, then expose, photograph, and store. 6.8. Experimental Results: As shown in Figure 1, recombinant adenovirus Ad5-ZIKV-E WT and Ad5-ZIKV-E mut Green fluorescence can be detected after infection of 293T cells because the recombinant adenovirus gene backbone carries a gene encoding green fluorescent protein (GFP). Figure 1 A). Furthermore, in Ad5-ZIKV-E WT and Ad5-ZIKV-E mut Zika virus E protein, with a molecular weight of approximately 55 kDa, can be detected in the supernatant of infected 293T cells. This is consistent with the theoretical molecular weight.

[0035] Example 7. Immunization of mice with recombinant adenovirus 7.1. Fifteen 6-week-old male C57BL / 6 mice were divided into 3 groups (Ad5-ZIKV-E). WT Group, Ad5-ZIKV-E mut Group 1 and PBS group, 5 animals in each group); 7.2. Subcutaneously inject 5×10⁷ vp Ad5-ZIKV-E into the back of each mouse. WT Or Ad5-ZIKV-E mut Alternatively, use the same volume of PBS as a control group; administer a booster immunization 14 days after immunization. 7.3. Two weeks after the second immunization, the mice were sacrificed, blood was collected from the heart and serum was separated. The serum was incubated in a 56°C water bath for 30 min to inactivate complement and stored at -20°C for later use.

[0036] Example 8. ELISA detection of antibodies in mouse serum 8.1. Coating Antigen: Add 20 μg / mL ZIKV-E410 (Zika virus E protein extracellular region, 1-410 aa) or JEV-E406 (Japanese encephalitis virus E protein extracellular region, 1-406 aa) or DENV2-E401 protein (dengue virus type 2 E protein extracellular region, 1-401 aa; diluted with coating buffer, 100 μL / well) to each well of a high-affinity ELISA plate and incubate at 37°C for 1.5 h. Discard the liquid in each well, add 200 μL of PBS to each well, shake for 10 seconds, and then discard the liquid in each well. 8.2. Sealing plate: Add 150 μL of 5% skim milk powder (dissolved in PBS) to each well and incubate at 37°C for 1.5 h. Discard the liquid in each well, add 200 μL of PBS to each well, shake for 10 seconds, and then discard the liquid in each well. 8.3. Add test serum: Add 100 μL of PBS-diluted mouse serum or recombinant adenovirus-immunized mouse serum (starting with a 1:30 dilution) to each well, and incubate at 37°C for 1 h. Discard the liquid from each well, and wash the plate 4 times with 0.5% PBST (PBS containing 0.5% Tween) (add 200 μL of 0.5% PBST to each well, shake for 10 seconds, and then discard the liquid from each well). 8.4. Add secondary antibody: Add 100 μL of HRP-labeled goat anti-mouse IgG secondary antibody (1:1000 dilution, diluted with PBS) to each well, incubate at 37°C for 1 h, discard the liquid from each well, and wash the plate 4 times with 0.5% PBST (add 200 μL of 0.5% PBST to each well, shake for 10 seconds, and then discard the liquid from each well). 8.5. Add colorimetric reagent: Add 40 μL of TMB colorimetric reagent (20 μL each of solution A and solution B) to each well, react at room temperature for 5 min, and add 20 μL of stop solution to each well; 8.6. OD value determination: The OD450 value of each well was determined using a microplate reader; 8.7. Experimental Results: As shown in A of Figure 2, compared to Ad5-ZIKV-E WT Ad5-ZIKV-E mut The ability of serum antibodies induced in immunized mice to recognize the Zika virus antigen (ZIKV-E410) was slightly reduced; as shown in Figures 2B and 2C, compared to Ad5-ZIKV-E WT Ad5-ZIKV-E mutThe ability of serum antibodies induced in immunized mice to cross-recognize Japanese encephalitis virus antigen (JEV-E406) and dengue virus antigen (DENV2-E401) was significantly weakened. Figure 2A shows the ELISA results coated with ZIKV-E410 protein, Figure 2B shows the ELISA results coated with JEV-E406 protein, and Figure 2C shows the ELISA results coated with DENV2-E401 protein.

[0037] Example 9. Virus neutralization experiment 9.1 Vero cells were seeded into 24-well cell culture plates (1×10⁵ / well) and cultured at 37°C in a 5% CO₂ incubator for 24 h; 9.2 The serum to be tested was serially diluted three times in a 96-well cell culture plate (240 μL / well, diluted with culture medium), and serum-free wells were set up (240 μL culture medium / well). ZIKV (50 PFU / 50 μL) was added to each well and incubated at 37°C for 1 h. The cell supernatant in the 24-well plate was discarded, and the virus-serum mixture was added to the cells in each well and infected at 37°C for 1 h. The virus solution was discarded, and 0.5 mL of 1% methylcellulose / culture medium was added to each well. The cells were incubated at 37°C in a 5% CO2 incubator for 4 days.

[0038] 9.3 Add 0.5 mL of 4% paraformaldehyde to each well and fix at room temperature for 1 h.

[0039] 9.4 Rinse the plate with tap water, add 0.2 mL of 0.5% crystal violet staining solution to each well, and stain at room temperature for 1 min.

[0040] 9.5 Wash the plates with tap water, allow them to air dry in 24-well plates, and count the number of viral plaques in each well. Calculate the percentage of virus neutralization by serum at different dilutions: Neutralization rate = [(Number of viral plaques in serum-free wells - Number of viral plaques in serum-free wells) / Number of viral plaques in serum-free wells] * 100% 9.6 The Prism 8 software was used to analyze the data, plot the graphs, and calculate the NT50 value (i.e., the highest serum dilution that neutralizes half of the virus).

[0041] 9.7 Experimental Results: As shown in Figure 3, Ad5-ZIKV-E WT Immunized mouse serum and Ad5-ZIKV-E mut The neutralizing capacity of immunized mouse serum for ZIKV was inversely proportional to its dilution. Ad5-ZIKV-E WT Immunized mouse serum and Ad5-ZIKV-E mutThe NT50 values ​​of ZIKV neutralization in immunized mouse serum were 1:199 and 1:112, respectively, with no significant difference between the two.

[0042] Example 10. In vitro ADE experiment 10.1 Seed K562 cells into 96-well U-shaped bottom cell culture plates (2×10⁴ / well) and incubate at 37°C in a 5% CO₂ incubator for 24 h; 10.2 The serum to be tested was serially diluted three times in a 96-well cell culture plate (50 μL / well, diluted with culture medium, starting from 1:10). Separate serum-free wells (50 μL PBS / well) and background wells (50 μL PBS / well) were also prepared. Except for the background wells, 50 μL of live JEV or DENV2 virus (containing 1000 PFU) was added to each of the other wells, and the plates were incubated at 37°C for 1 h. 10.3 Centrifuge the K562 cell culture plate at 2000 rpm for 10 min, discard the supernatant from each well, add the mixed liquid to each well of a standard 96-well plate to the K562 cells, incubate at 37℃ for 1 h, centrifuge the K562 cell culture plate at 2000 rpm for 10 min, discard the liquid from each well, and wash each well once with PBS. Add 150 μL of RPMI-1640 complete culture medium to each well, and incubate at 37℃ in a 5% CO2 incubator for 48 h; 10.4 Centrifuge K562 cell culture plates at 2000 rpm for 10 min, collect the supernatant from each well, and determine the viral titer using a plaque formation assay (PFA). The experimental steps are as follows: A. Seed Vero cells in 24-well cell culture plates (1×10⁵ / well) and incubate at 37°C in a 5% CO₂ incubator for 24 h; B. In a 96-well cell culture plate, serially dilute the supernatant of the cells to be tested three times (240 μL / well, diluted with culture medium, starting with a 1:3 ratio). Discard the cell supernatant in the 24-well plate, add the cell supernatant of different dilutions to the cells, and infect at 37°C for 1 h.

[0043] C. Discard the virus solution, add 0.5 mL of 1% methylcellulose / culture medium to each well, and incubate at 37°C in a 5% CO2 incubator for 3 days.

[0044] D. Add 0.5 mL of 4% paraformaldehyde to each well and fix at room temperature for 1 h.

[0045] E. Wash the plate with tap water, add 0.2 mL of 0.5% crystal violet staining solution to each well, and stain at room temperature for 1 min.

[0046] F. Wash the plate with tap water, air dry the 24-well plate, count the number of viral plaques in each well, and express the viral titer in the cell supernatant as PFU / mL.

[0047] G. The formula for calculating the fold increase in JEV or DENV2 infection by different serum dilutions is: fold increase in infection = (virus titer in cell supernatant of different serum dilutions - virus titer in cell supernatant without serum) / virus titer in cell supernatant without serum.

[0048] 10.5 Experimental Results: Regarding JEV infection of K562 cells in vitro, Ad5-ZIKV-E WT Or Ad5-ZIKV-E mut The enhancing effect of immunized mouse serum on JEV infection initially increased and then decreased with increasing serum dilution. Both sera showed the greatest enhancing effect on JEV infection at a 90-fold dilution (1:90). Ad5-ZIKV-E mut The serum of immunized mice enhanced JEV infection by 38-fold compared to Ad5-ZIKV-E. WT The effect of immune mouse serum on JEV-enhancing infection (which was enhanced by 189-fold) was reduced by 151-fold (Figure 4A).

[0049] Similarly, Ad5-ZIKV-E WT Or Ad5-ZIKV-E mut The enhancing effect of immunized mouse serum on DENV2 infection initially increased and then decreased with increasing serum dilution. Ad5-ZIKV-E WT The enhanced effect of immunized mouse serum on DENV2 infection was greatest (46-fold enhancement) at a 30-fold dilution (1:30), while Ad5-ZIKV-E... mut The enhanced effect of immunized mouse serum on DENV2 infection was greatest (10-fold enhancement) at a 90-fold dilution (1:90). Therefore, Ad5-ZIKV-E mut The enhancing effect of immunized mouse serum on DENV2 infection was greater than that of Ad5-ZIKV-E. WT The effect of immunized mouse serum on DENV2-enhanced infection was reduced by 36-fold (Figure 4B).

[0050] Example 11. Neutralizing effect of adoptive immune serum on ZIKV infection in mice. 11.1 Add 10 μL of PBS or Ad5-ZIKV-E WT Immunized mouse serum or Ad5-ZIKV-E mut Serum from immunized mice was injected subcutaneously into 7-day-old AB6 mice.

[0051] 11.2 Two hours later, 100 PFU of ZIKV was injected subcutaneously into the mice.

[0052] 11.3 Four days after ZIKV infection, mice were sacrificed, and mouse serum and brain tissue were collected. The viral load in mouse serum and brain tissue was determined by PFA (experimental method as above).

[0053] 11.4 The ZIKV load in mouse serum and brain tissue is expressed as PFU / mL and PFU / g, respectively.

[0054] 11.5 Use Prism software to analyze the data and create graphs.

[0055] 11.6 Experimental Results: Ad5-ZIKV-E was given WT Immunized mouse serum or Ad5-ZIKV-E mut Three days after ZIKV infection, the ZIKV viral titer in the serum of AB6 mice immunized with mouse serum was significantly lower than that in the serum of AB6 mice given serum from unimmunized mice (126 PFU / mL). versus 50118 PFU / mL, p<0.0001; 229 PFU / mL versus 50118 PFU / mL, p<0.0001 (Figure 5A). Furthermore, Ad5-ZIKV-E was injected. WT Immunized mouse serum or Ad5-ZIKV-E mut Virus was undetectable in the brain tissue of AB6 mice from immunized mouse serum (viral titer at the detection limit), which was significantly lower than the viral load in the brain tissue of control mice (4 PFU / g). versus 54 PFU / g, p<0.0001; 4 PFU / g versus 54 PFU / g, p<0.0001 (Figure 5B). Importantly, the injection of Ad5-ZIKV-E... WT Mice in the immunized mouse serum group and those injected with Ad5-ZIKV-E mut There were no significant differences in serum among the immunized mice.

[0056] Example 12. Enhancement of DENV2 infection in mice by adoptive immune serum. 12.1 Add 10 μL of PBS or Ad5-ZIKV-E WT Immunized mouse serum or Ad5-ZIKV-E mut Serum from immunized mice was injected subcutaneously into 7-day-old AB6 mice.

[0057] 12.2 Two hours later, 100 PFU of DENV2 was injected subcutaneously into the mice.

[0058] 12.3 Four days after DENV2 infection, mice were sacrificed, and mouse serum and brain tissue were collected. The viral load in mouse serum and brain tissue was determined by PFA (experimental method as above).

[0059] 12.4 The DENV2 load in mouse serum and brain tissue is expressed as PFU / mL and PFU / g, respectively.

[0060] 12.5 Use Prism software to analyze the data and create graphs.

[0061] 12.6 Experimental Results: Injected Ad5-ZIKV-E WT Three days after DENV2 infection, the viral titer in the serum of AB6 mice immunized with mouse serum was significantly higher than that in the serum of control mice (89125 PFU / mL). versus The viral load in the brain tissue of mice was 2454 PFU / mL, p<0.05, which is the ADE phenomenon (Figure 6A). Furthermore, the viral load in the brain tissue of these mice was also higher than that in the brain tissue of the control group (501 PFU / g). versus 131 PFU / g (Figure 6B). Then, Ad5-ZIKV-E was injected. mut AB6 mice immunized with serum from immunized mice showed lower viral loads in serum and brain tissue three days after DENV2 infection compared to mice injected with Ad5-ZIKV-E. WT The viral load in AB6 mouse serum and brain tissue of immunized mice was 8912 PFU / mL. versus 89125 PFU / mL, 117 PFU / g versus 501 PFU / g (Figure 6).

[0062] Example 13. Enhancement effect of maternally acquired antibodies in suckling mice on JEV infection. 12.1 Ad5-ZIKV-E WT Or Ad5-ZIKV-E mut Female C57BL / 6 mice were immunized (using the same method as above). Mice immunized with PBS served as controls.

[0063] 12.2 The above-immunized female mice were paired with male mice of the same age.

[0064] 12.3 After the above-mentioned pregnant mice gave birth to 1-day-old suckling mice, 100 PFU of JEV was injected subcutaneously into the 1-day-old suckling mice.

[0065] 12.4 Three days later, the suckling mice were euthanized and serum and brain tissue were collected. The JEV load in the serum and brain tissue of the suckling mice was determined by PFA (method as above).

[0066] 12.5 Viral load in the serum and brain tissue of suckling mice is expressed as PFU / mL.

[0067] 12.6 Use Prism 8 software to analyze the data and create graphs.

[0068] 12.7 Experimental Results: Ad5-ZIKV-E WT One-day-old suckling mice born to immunized mice had significantly higher viral titers in their serum three days after JEV infection than one-day-old suckling mice born to unimmunized mice (44,668,359 PFU / mL). versus 15135612 PFU / mL, p<0.05). Ad5-ZIKV-E mut One-day-old suckling mice born to immunized mice showed significantly lower viral titers in their serum after JEV infection compared to control mice, and also significantly lower than those in Ad5-ZIKV-E mice. WT Viral titers in the serum of suckling mice born to immunized mice were 3,467,368 PFU / mL. versus 44668359 PFU / mL, p<0.0001; 3467368 PFU / mL versus 15135612 PFU / mL, p<0.01 (Figure 7).

[0069] Unless otherwise defined, all terms, symbols, and other scientific terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In some instances, terms having a conventional meaning are defined herein for clarification or ease of reference, and such definitions should not be construed as indicating a significant difference from conventional understanding in the art. The technical methods described or referenced herein are generally well understood by those skilled in the art and employed by conventional methods. Unless otherwise stated, the use of commercially available kits, reagents, and instruments shall be performed according to the manufacturer's instructions and parameters.

[0070] While the disclosure is as stated above, its scope of protection is not limited thereto. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of this disclosure, and all such changes and modifications will fall within the protection scope of this invention.

Claims

1. A Zika virus recombinant adenovirus shuttle plasmid, characterized in that, The Zika virus recombinant adenovirus shuttle plasmid carries the Zika virus E protein mutant gene, and the amino acid sequence encoded by the Zika virus E protein mutant gene is shown in SEQ ID No.

3.

2. The Zika virus recombinant adenovirus shuttle plasmid as described in claim 1, characterized in that, The nucleotide sequence of the Zika virus E protein mutant gene is shown in SEQ ID No.

4.

3. The Zika virus recombinant adenovirus shuttle plasmid as described in claim 1 or 2, characterized in that, The 5' end of the Zika virus E protein mutant gene is linked to a signal peptide gene, the 5' end of which is connected to a Kozak sequence, and the 5' end of the Kozak sequence is connected to an enzyme cleavage site. The amino acid sequence encoded by the signal peptide gene is shown in SEQ ID No. 5, and the nucleotide sequence of the signal peptide gene is shown in SEQ ID No.

6. The specific nucleotide sequence of the Kozak sequence is gccacc, and the specific nucleotide sequence of the enzyme cleavage site is gaattc.

4. The Zika virus recombinant adenovirus shuttle plasmid as described in claim 1 or 2, characterized in that, The 3' end of the Zika virus E protein mutant gene is connected to a second restriction enzyme site, the specific nucleotide sequence of which is ctcgag.

5. The Zika virus recombinant adenovirus shuttle plasmid as described in claim 1 or 2, characterized in that, The vector for the Zika virus recombinant adenovirus shuttle plasmid is pShuttle.

6. A method for preparing a Zika virus recombinant adenovirus shuttle plasmid, characterized in that, Includes the following steps: S1: The pShuttle plasmid and the Zika virus E protein mutant gene were double-digested using restriction endonucleases EcoRI and Xhol. S2: Perform 0.8% agarose gel electrophoresis on the enzyme digestion products of step S1, and cut out the bands of the target gene and pShuttle respectively. Use an agarose gel purification and recovery kit to recover pShuttle and the target gene Zika virus E protein mutant gene. S3: Use T4 DNA ligase to ligate pShuttle with the Zika virus E protein mutant gene; S4: Add the reaction system after step S3 to Stbl3 competent cells, gently mix, incubate on ice for 30 min, incubate in water at 42℃ for 90 s, quickly transfer to ice and incubate on ice for 2 min, add antibiotic-free LB culture medium to the cells, and incubate with shaking at 180 rpm / 37℃ for 1 h. S5: Centrifuge, remove the supernatant, mix the remaining bacterial culture and inoculate it onto LB agar medium containing 50 μg / mL kanamycin. Incubate at 37°C upright until the liquid is absorbed, then invert and incubate at 37°C overnight. S6: Select white bacterial clones and add them to 10 mL of LB medium containing 50 μg / mL kanamycin, and incubate with shaking at 220 rpm / 37℃ for 18 h; S7: Extract recombinant plasmids from bacterial cultures using a plasmid mini-extraction kit and determine the plasmid concentration.

7. A Zika virus recombinant adenovirus plasmid, characterized in that, The plasmid carries the exogenous Zika virus E protein mutant gene, which is obtained by transforming the Zika virus recombinant adenovirus shuttle plasmid as described in any one of claims 1 to 5 and the human adenovirus type 5 genome backbone plasmid pAdEasy-1 into Escherichia coli BJ5183 for homologous recombination and then screening.

8. A Zika virus recombinant adenovirus, characterized in that, It is obtained by transfecting the Zika virus recombinant adenovirus plasmid as described in claim 7 into a host cell.

9. The use of the Zika virus recombinant adenovirus as described in claim 8 in the preparation of a Zika virus recombinant adenovirus vaccine.

10. The application as described in claim 9, characterized in that, The process includes the following steps: inoculating the Zika virus recombinant adenovirus of claim 8 into host cells and then passaged them continuously, and after the transcription and expression of the recombinant adenovirus E protein in the host cells are stabilized, a Zika virus recombinant adenovirus vaccine is obtained.

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